Milk Basic Protein Facilitates Increased Bone Mass in Growing Mice.

Milk basic protein (MBP) comprises a group of basic whey proteins and is effective in preventing bone loss by promoting bone deposition (bone formation) and suppressing withdrawn (bone resorption). We previously revealed the bone protective effects of MBP during life phases involving excessive bone resorption, such as in adults and postmenopausal women, and in animal models (ovariectomized rats and mice). However, it was unclear whether MBP increases bone mass during the growth stage, when there is more bone formation than resorption. We therefore investigated the effect of MBP supplementation on bone mass in 6-wk-old mice provided water supplemented with MBP [0.01%, 0.1%, 1.0% (w/w)] or deionized water (control) ad libitum for 10 wk. Analysis by micro-computerized tomography showed that MBP significantly increased tibia cortical bone mineral density and femur trabecular bone volume to tissue volume compared with mice provided deionized water. Next, the function of MBP in bone remodeling (bone formation and resorption) was evaluated using an in vitro system and the results demonstrated that MBP directly promoted osteoblast proliferation and inhibited osteoclastogenesis. Moreover, the plasma level of insulin-like growth factor-1 was increased by MBP supplementation, suggesting that MBP indirectly promoted osteoblast proliferation/differentiation. These effects enhance bone formation and/or inhibit bone resorption, resulting in increased bone mass in growing mice.

Milk basic protein increases ghrelin secretion and bone mineral density in rodents.

OBJECTIVES: Milk basic protein (MBP), a mixture of proteins isolated from bovine milk, is known to increase bone formation. Ghrelin, a stomach-derived peptide hormone, also has been reported to stimulate osteoblast formation. The aim of this study was to determine whether MBP-induced bone formation is mediated via ghrelin. METHODS: MBP was chronically administered to mice in their drinking water for 3 wk, and body weight, water intake, and bone mineral density were measured. Additionally, plasma bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase isoform 5b, and ghrelin concentrations were determined by enzyme-linked immunosorbent assay. To examine the direct effect of MBP on ghrelin secretion, gastric tissue culture and primary mucosal cells were stimulated by MBP. RESULTS: The in vivo study of young, growing mice showed that chronic MBP intake for 3 wk increased the plasma ghrelin concentration and bone mineral density of the hind limb tibia. In vitro studies using minced rat gastric mucosa tissues and primary murine isolated gastric mucosal cells revealed that MBP stimulated ghrelin release in a dose-dependent manner. Moreover, MBP-induced ghrelin secretion was partly inhibited by adrenergic blockers. CONCLUSIONS: These findings suggest a novel mechanism by which MBP directly acts on ghrelin secretion. Additionally, the elevated ghrelin level induced by MBP may act as a mediator for bone formation.

Modulation of brown adipocyte activity by milk by-products: Stimulation of brown adipogenesis by buttermilk.

Brown adipocytes dissipate chemical energy in the form of heat through the expression of mitochondrial uncoupling protein 1 (Ucp1); Ucp1 expression is further upregulated by the stimulation of ß-adrenergic receptors in brown adipocytes. An increase in energy expenditure by activated brown adipocytes potentially contributes to the prevention of or therapeutics for obesity. The present study examined the effects of milk by-products, buttermilk and butter oil, on brown adipogenesis and the function of brown adipocytes. The treatment with buttermilk modulated brown adipogenesis, depending on the product tested; during brown adipogenesis, buttermilk 1 inhibited the differentiation of HB2 brown preadipocytes. In contrast, buttermilk 3 and 5 increased the expression of Ucp1 in the absence of isoproterenol (Iso), a ß-adrenergic receptor agonist, suggesting the stimulation of brown adipogenesis. In addition, the Iso-induced expression of Ucp1 was enhanced by buttermilk 2 and 3. The treatment with buttermilk did not affect the basal or induced expression of Ucp1 by Iso in HB2 brown adipocytes, except for buttermilk 5, which increased the basal expression of Ucp1. Conversely, butter oil did not significantly affect the expression of Ucp1, irrespective of the cell phase of HB2 cells, ie, treatment during brown adipogenesis or of brown adipocytes. The results of the present study indicate that buttermilk is a regulator of brown adipogenesis and suggest its usefulness as a potential food material for antiobesity.

A liquid-helium-free superconducting coil system forming a flat minimum-magnetic-field distribution of an electron cyclotron resonance ion source.

A flat distribution of the minimum magnetic field (flat-Bmin) of an electron cyclotron resonance ion source (ECRIS) is expected to perform better in highly charged ion production than classical Bmin. To form a flat-Bmin structure with a liquid helium-free superconducting device, a coil system of seven coils with four current leads has been designed. The lead number was reduced by connecting the plural coils in series to maintain the flat-Bmin structure even when the coil currents are changed for adjustment. This coil system can be operated with a helium-free cryostat, since the estimation of heat from the leads to the coils is nearly equivalent to the existing superconducting ECRIS of a similar type.

Induction of beige-like adipocytes in 3T3-L1 cells.

There are two types of brown adipocytes: classical brown adipocytes that form the brown fat depots and beige adipocytes that emerge in the white fat depots. Beige adipocytes have a low level of uncoupling protein 1 (Ucp1) expression in the basal state, but Ucp1 expression is increased in response to ß adrenergic receptor activation. The present study explored the factors responsible for the differentiation of 3T3-L1 white preadipocytes to beige adipocytes. Significant expression of Ucp1 was not detected under any tested conditions in the absence of isoproterenol (Iso), an agonist of ß adrenergic receptor. Iso-induced Ucp1 expression was significantly higher in the cells treated with a mixture of triiodothyronine (T3) and 3-isobutyl-1-methylxanthine (IBMX) for days 0-8 than in the control cells. Chronic IBMX treatment was indispensable for the enhanced Iso-induced Ucp1 expression, and treatment with additional rosiglitazone (Rosi) for days 0-8 further increased the Ucp1 expression. Recently, genes were identified that are predominantly expressed in beige adipocytes, which were induced from stromal vascular cells in white fat depots. However, the expression levels of the beige adipocyte-selective genes in the adipocytes induced by the mixture of T3, IBMX and Rosi did not differ from those in the control adipocytes. The present study indicates that 3T3-L1 cells can differentiate to beige-like adipocytes by prolonged treatment with the mixture of T3, IBMX and Rosi and that the gene expression profile of the adipocytes is distinct from those previously induced from white fat depots.

Cytochemistry of sialoglycoconjugates and lysozyme in canine anal glands as studied by electron microscopic methods.

The distribution of sialoglycoconjugates and lysozyme in the secretory cells of canine anal glands was studied by means of electron microscopic cytochemical methods, particularly lectin cytochemistry and immunocytochemistry. Sialic acids were predominantly present in the secretory granules, Golgi bodies, surface coat of the plasma membrane and luminal secretions. In addition, within these structures, the secretory granules, Golgi bodies and luminal secretions exhibited high levels of sialoglycoconjugates that terminated in Siaα2-6Gal/GalNAc or Siaα2-3Galß1-4GlcNAc. In the secretory cells, reactive gold particles representing lysozyme were mainly detectable in the secretory granules and Golgi bodies. Sialic acids possess diverging functional properties, whereas lysozyme contributes to the non-specific defense against microorganisms. Therefore, their presence and secretion are suggestive of protective effects of both secretory products at the anal mucosa.

Sialic acids and antimicrobial substances in the apocrine glands of porcine perianal skin.

The porcine perianal skin shows prominent apocrine glands with large saccular dilatations, whereby the functional significance of the glandular secretions is rather unexplained. Our study focuses on the demonstration of sialoglycoconjugates and antimicrobial substances in these glands, using glycoconjugate histochemical and immunohistochemical methods. The result obtained emphasized the general presence of sialic acids, linked to α2-6Gal/GalNAc and α2-3Gaßl1-4GlcNAc, in the secretory cells. The secretory epithelium and luminal secretions also contained a spectrum of antimicrobial substances, such as lysozyme, IgA, lactoferrin, and the peptide group of ß-defensins. Realizing that sialic acids possess diverging functional properties through various saccharide residues, and that antimicrobial substances serve as a non-specific defense against microorganisms, these secretory products may function as protective agents in order to preserve the integrity of the perianal region. This view includes that the amounts of bacteria on the skin surface are controlled and maintained at the certain level.

Histochemical demonstration of sialic acids and antimicrobial substances in the porcine anal glands.

The distribution of sialic acids and antimicrobial products (lysozyme, ß-defensin-1, lactoferrin, IgA) in the anal glands of miniature pig was studied by glycoconjugate histochemistry and immunohistochemistry. The glandular acini of these glands exhibited considerable amounts of sialoglycoconjugates that terminated in Siaα2-6Gal/GalNAc or Siaα2-3Gal1-4GlcNAc, including O-acetylated sialic acids. Additionally, all antimicrobial products examined could be demonstrated in the anal glands, especially in the serous cells. The results obtained are discussed with regard to the functional significance of the anal glands. Our observations corroborated the view that sialic acids closely interact with defense cells and antimicrobial substances in the innate immune response. Therefore, the anal glandular secretions may function as protective agents in order to preserve the integrity of the anal region.

Functional properties of feline foot pads as studied by lectin histochemical and immunohistochemical methods.

The localization of sialic acids and antimicrobial substances in the foot pads of the cat was examined by lectin histochemical and immunohistochemical methods. The lectin binding patterns of the eccrine glands were suggestive of the existence of large concentrations of sialoglycoconjugates that terminated in Siaalpha2-3Gal1-4GlcNAc. Results were consistent with localization of O-linked (mucin-type) sialoglycoproteins with the Siaalpha2-6Gal/GalNAc sequence in the epidermal layers, especially the stratum spinosum. Additionally, antimicrobial peptides, such as lysozyme, secretory component, lactoferrin, and the peptide group of beta-defensins were demonstrated to be immunolocalised in the eccrine glandular cells. These substances, except for secretory component, were also distributed in the epidermal strata. The sialic acids and antimicrobial substances found in the eccrine glandular secretions and epidermis may play an essential role in the preservation of skin integrity in feline foot pads.

DNA polymerase mu interacts with a meiosis-specific RecA homolog Lim15 during meiosis in Coprinus cinereus.

Meiosis is a fundamental process in eukaryotes. Homologous chromosomes are paired and recombined during meiotic prophase I, which results in variation among the gametes. However, the mechanism of recombination between the maternal and paternal chromosome is unknown. In this study, we report on the identification of interaction between Coprinus cinereus DNA polymerase mu (CcPol mu) and CcLim15/Dmc1, a meiosis-specific RecA-like protein, during meiosis. Interaction between these two proteins was confirmed using a GST-pull down assay. A two-hybrid assay revealed that the N-terminus of CcPol mu, which includes the BRCT domain, is responsible for binding the C-terminus of CcLim15. Furthermore, co-immunoprecipitation experiments indicate that these two proteins also interact in the crude extract of the meiotic cell. A significant proportion of CcPol mu and CcLim15 is shown to co-localize in nuclei from the leptotene/zygotene stage to the early pachytene stage during meiotic prophase I. Moreover, CcLim15 enhances polymerase activity of CcPol mu early in the reaction. These results suggest that CcPol mu might be recruited by CcLim15 and elongate the D-loop structure during homologous recombination in meiosis.

High-throughput protein refolding screening method using zeolite.

We established a 96-well-plate-based refolding screening system using zeolite. In this system, protein denatured and solubilized with 6 M guanidine hydrochloride is adsorbed onto zeolite placed in a 96-well plate. The refolding conditions can be tested by incubating the samples with refolding buffers under various conditions of pH, salts, and additives. In this study, we chose green fluorescent protein as the model protein. Green fluorescent protein was expressed as inclusion bodies, and we tested the effects of four pH conditions and six additives on its refolding. The results demonstrate that green fluorescent protein was more efficiently refolded with zeolite than with the conventional dilution method.

Single-turn extraction from a K110 AVF cyclotron by flat-top acceleration.

Single-turn extraction from the Japan Atomic Energy Agency AVF cyclotron with a K number of 110 using a flat-top (FT) acceleration system has been achieved to reduce the energy spread of an ion beam for microbeam formation with energy up to hundreds of MeV and to increase extraction efficiency from the cyclotron. In order to generate a FT waveform voltage using the fifth-harmonic frequency on a dee electrode, a FT resonator was designed using MAFIA code to achieve downsizing and low power consumption. The FT resonator, coupled to the main resonator through a coupling capacitor, covered the full range of the fifth harmonic frequency from 55 to 110 MHz. Various ion beams, accelerated using different acceleration harmonic modes of h=1 and 2, such as 220 MeV (12)C(5+) (h=2), 260 MeV (20)Ne(7+) (h=2), and 45 MeV H(+) (h=1), were developed by FT acceleration. A clear turn separation of the beam bunches was successfully observed at the extraction region of the large-scale AVF cyclotron with number of revolutions greater than 200. As a result, high extraction efficiency (over 95%) from the cyclotron was achieved. Single-turn extraction was confirmed by counting the number of beam bunches out of the cyclotron for an injected beam pulsed by a beam chopping system in the injection line. The energy spread of the 260 MeV (20)Ne(7+) beam was measured using an analyzing magnet, and we verified a reduction in the energy spread from DeltaE/E=0.1% to 0.05% by single-turn extraction after FT acceleration.

Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (-/-), which enabled us to study HUFA deficiency without depleting their precursors. In -/-, no in vivo AA synthesis was detected after administration of [U-(13)C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the -/- developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The -/- also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male -/- lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in -/- declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in -/-. DPAn-6 was absent in -/- brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (-/-) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.

Refolding of lactate dehydrogenase by zeolite beta.

We used zeolite beta as an adsorbing matrix to refold recombinant lactate dehydrogenase (LDH) protein collected as an insoluble aggregate from a bacterial expression system. The adsorption isotherm revealed that 1 g of zeolite adsorbed 200 mg of denatured LDH solubilized with a buffer containing 6 M of guanidine hydrochloride. The pH of the buffer had little effect on the adsorption, but this property was abolished by preincubation of the zeolite with polyethylene glycol (PEG) in a weight ratio of 1:10. These data suggest that the adsorption of LDH depends on the hydrophobicity of the zeolite surface, and that the adsorption of PEG to zeolite is sufficient to release LDH from its surface. LDH was thus released by refolding buffer containing PEG and arginine, and soluble LDH was obtained in its active enzymatic form. The addition of arginine dramatically increased the yield of LDH in a dose-dependent manner. The overall refolding efficiency was optimized to 35%.

Use of zeolite to refold a disulfide-bonded protein.

Zeolites are microporous crystalline aluminosilicates with a highly ordered structure. Using zeolite beta as an adsorbent, denatured/reduced hen egg lysozyme was refolded to the active form at high concentrations. The denatured/reduced lysozyme was adsorbed onto the zeolite and the protein was refolded by desorbing it into refolding buffer, consisting of redox reagents, guanidine hydrochloride, polyethylene glycol, and L-arginine. This zeolite refolding method could be highly effective for various kinds of proteins, refolding them with high efficiency even when they contain disulfide bonds.

Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1: a molecular link between recombination and chromatin assembly during meiosis.

In eukaryotes, meiosis leads to genetically variable gametes through recombination between homologous chromosomes of maternal and paternal origin. Chromatin organization following meiotic recombination is critical to ensure the correct segregation of homologous chromosomes into gametes. However, the mechanism of chromatin organization after meiotic recombination is unknown. In this study we report that the meiosis-specific recombinase Lim15/Dmc1 interacts with the homologue of the largest subunit of chromatin assembly factor 1 (CAF-1) in the basidiomycete Coprinopsis cinerea (Coprinus cinereus). Using C. cinerea LIM15/DMC1 (CcLIM15) as the bait in a yeast two-hybrid screen, we have isolated the C. cinerea homologue of Cac1, the largest subunit of CAF-1 in Saccharomyces cerevisiae, and named it C. cinerea Cac1-like (CcCac1L). Two-hybrid assays confirmed that CcCac1L binds CcLim15 in vivo. beta-Galactosidase assays revealed that the N-terminus of CcCac1L preferentially interacts with CcLim15. Co-immunoprecipitation experiments showed that these proteins also interact in the crude extract of meiotic cells. Furthermore, we demonstrate that, during meiosis, CcCac1L interacts with proliferating cell nuclear antigen (PCNA), a component of the DNA synthesis machinery recently reported as an interacting partner of Lim15/Dmc1. Taken together, these results suggest a novel role of the CAF-1-PCNA complex in meiotic events. We propose that the CAF-1-PCNA complex modulates chromatin assembly following meiotic recombination.

Dietary fructose induces a wide range of genes with distinct shift in carbohydrate and lipid metabolism in fed and fasted rat liver.

Dietary fructose has been suspected to contribute to development of metabolic syndrome. However, underlying mechanisms of fructose effects are not well characterized. We investigated metabolic outcomes and hepatic expression of key regulatory genes upon fructose feeding under well defined conditions. Rats were fed a 63% (w/w) glucose or fructose diet for 4 h/day for 2 weeks, and were killed after feeding or 24-hour fasting. Liver glycogen was higher in the fructose-fed rats, indicating robust conversion of fructose to glycogen through gluconeogenesis despite simultaneous induction of genes for de novo lipogenesis and increased liver triglycerides. Fructose feeding increased mRNA of previously unidentified genes involved in macronutrient metabolism including fructokinase, aldolase B, phosphofructokinase-1, fructose-1,6-bisphosphatase and carbohydrate response element binding protein (ChREBP). Activity of glucose-6-phosphate dehydrogenase, a key enzyme for ChREBP activation, remained elevated in both fed and fasted fructose groups. In the fasted liver, the fructose group showed lower non-esterified fatty acids, triglycerides and microsomal triglyceride transfer protein mRNA, suggesting low VLDL synthesis even though plasma VLDL triglycerides were higher. In conclusion, fructose feeding induced a broader range of genes than previously identified with simultaneous increase in glycogen and triglycerides in liver. The induction may be in part mediated by ChREBP.

Immunocytochemical localization of lysozyme and beta-defensin in the apocrine glands of the equine scrotum.

The present study revealed in detail the subcellular localization of lysozyme and beta-defensin in the apocrine glands of the equine scrotal skin, a specific body region. The apocrine glandular cells were equipped with a varying number of secretory granules, a well-developed Golgi apparatus and abundant cisternae of the rough endoplasmic reticulum within their cytoplasm. In these cells, reactive gold particles representing lysozyme were detectable in the secretory granules as well as the Golgi apparatus and elements of the rough endoplasmic reticulum. Additionally, the antimicrobial peptide group of beta-defensin was also localized in the above-mentioned ultrastructures of the secretory cells. The presence and secretion of such substances that may serve as a non-specific defense against microorganisms are suggestive of the protective effect of the secretory production elaborated by the apocrine glands.

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity.

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.

Dietary lycopene downregulates carotenoid 15,15'-monooxygenase and PPAR-gamma in selected rat tissues.

In vitro studies have suggested that lycopene is an efficient substrate for carotenoid 9'10'-monooxygenase II (CMO2) but an inhibitor of carotenoid 15,15'-monooxygenase I (CMO1). The objectives of this study were to clone the rat CMO2 gene, determine whether feeding lycopene for different lengths of time (3-37 d) altered the expression of genes related to carotenoid cleavage [CMO1, CMO2 and peroxisomal proliferator-activated receptor gamma (PPAR-gamma)] or increased the activity of selected phase I and phase II detoxification enzymes in rat tissues. The cloned rat CMO2 gene was 92 and 82% homologous to the mouse and human CMO2 nucleotide sequence, respectively. The relative abundance of CMO1, CMO2, and PPAR-gamma were differentially expressed among rat tissues. CMO1 and PPAR-gamma expression were decreased in the kidney and adrenal with lycopene intake (P < 0.05), whereas CMO2 expression was reduced only in the kidney. Lycopene did not alter hepatic phase I activity, but hepatic quinone reductase activity increased after 3 and 7 d of lycopene feeding (P < 0.05). Lycopene intake decreased a PPAR-gamma target gene, fatty acid binding protein 3 (FABP3), in the kidney and adrenal (P < 0.05). Thus, these data show that although the intake of 0.25 g lycopene/kg diet does not induce hepatic P450 detoxification enzymes, lycopene feeding alters CMO1, PPAR-gamma, and FABP3 mRNA expression in selected rat tissues with a moderate effect on kidney CMO2 expression. These data suggest that lycopene may play an important role in the modulation of beta-carotene, retinoid, and/or lipid metabolism.